Convenient Synthesis of an N-Glycan Octasaccharide of the Bisecting

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Convenient Synthesis of an N-Glycan Octasaccharide of the Bisecting Type Guangfa Wang, Wei Zhang, Zhichao Lu, Peng Wang, Xiuli Zhang, and Yingxia Li* Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean UniVersity of China, Qingdao 266003, China [email protected] ReceiVed January 5, 2009

Convenient synthesis of an N-glycan octasaccharide (A) of the bisecting type was described. The stable and easily prepared orthoester F, 3′′-O-chloroacetyl-4′′,6′′-O-benzylidene-R-D-glucopyranose 1′′,2′′(chloromethyl 3′,6′-di-O-benzyl-2′-deoxy-2′-phthalimido-β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2deoxy-2-phthalimido-β-D-glucopyranosylazide-4′-yl orthoacetate), was designed as the key intermediate with two advantages: (1) distinguishing OH-2′′ from OH-3′′ in β-D-glucopyranoside to construct the central β-D-mannopyranoside by inverting the configuration of OH-2′′ in β-D-glucopyranoside and (2) distinguishing OH-4′′ and OH-6′′ from OH-3′′ in the β-D-mannopyranoside to introduce the bisecting GlcNAc residue.

Introduction Glycoproteins with N-glycans found on cell surfaces and in blood serum play important roles in many key biological events. It is widely recognized that the functions of glycoproteins are influenced by their N-glycans.1 Among the great variety of their functions, tumor metastasis is a focal point in scientific research nowadays. β-1f6 GlcNAc branching, produced by GnT-V, plays a major role in cancer invasion and metastasis by stabilizing the proteases and angiogenic releasing factor.2 While (1) (a) Dwek, R. A. Science 1995, 269, 1234. (b) Dwek, R. A. Chem. ReV. 1996, 96, 683. (c) Van Dijk, W.; Van der Stelt, M. E.; Salera, A.; Dente, L. Eur. J. Cell Biol. 1991, 55, 143. (d) Shevinsky, L. H.; Knowles, B. B.; Damjanov, I.; Solter, D. Cell 1982, 30, 697. (e) Feizi, T. Nature 1985, 314, 53. (2) (a) Dennis, J. W.; Laferte, S.; Waghorne, C.; Breitman, M. L.; Kerbel, R. S. Science 1987, 236, 582. (b) Zhao, Y.; Nakagawa, T.; Itoh, S.; Inamori, K.; Isaji, T.; Kariya, Y.; Kondo, A.; Miyoshi, E.; Miyazaki, K.; Kawasaki, N.; Taniguchi, N.; Gu, J. J. Biol. Chem. 2006, 281, 32122.

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bisected N-glycans, which are the product of GnT-III, have unique function in terms of the suppression of cancer metastasis,3 they also have been shown to be involved in cell development4 and function of receptors.5 Therefore, the bisected N-glycans are of great significance with regard to cancer therapy. Because of difficulties in obtaining homogeneous glycans from natural sources, we decided to synthesize octasaccharide A, which is a part of the structure of a bisected N-glycan and (3) (a) Narasimhan, S. J. Biol. Chem. 1982, 257, 10235. (b) Iijima, J.; Zhao, Y.; Isaji, T.; Kameyama, A.; Nakaya, S.; Wang, X. C.; Ihara, H.; Cheng, X.; Nakagawa, T.; Miyoshi, E.; Kondo, A.; Narimatsu, H.; Taniguchi, N.; Gu, J. J. Biol. Chem. 2006, 281, 13038. (c) Zhao, Y.; Sato, Y.; Isaji, T.; Fukuda, T.; Matsumoto, A.; Miyoshi, E.; Gu, J.; Taniguchi, N. FEBS J. 2008, 275, 1939. (4) Yoshimura, M.; Ihara, Y.; Nishiura, T.; Okajima, Y.; Ogawa, M.; Yoshida, H.; Suzuki, M.; Yamamura, K. I.; Kanakura, Y.; Matsuzawa, Y.; Taniguchi, N. Biochem. J. 1998, 331, 733. (5) Ihara, Y.; Sakamoto, Y.; Mihara, M.; Shimizu, K.; Taniguchi, N. J. Biol. Chem. 1997, 272, 9629.

10.1021/jo900016j CCC: $40.75  2009 American Chemical Society Published on Web 02/26/2009

Synthesis of an N-Glycan Octasaccharide

FIGURE 1. Retrosynthesis of octasaccharide A.

suitable for enzymatic elongation and coupling to proteins for function study. However, the synthesis of bisected N-glycans is very difficult due to the sterically crowded β-mannosyl center. Only a few groups have developed synthetic approaches to this important class of compounds.6 Among them, Unverzagt’s strategy for introduction of the bisecting GlcNAc residue has been proven the most effective.6 In fact, the construction of the central β-Dmannosidic linkage with proper protection is the other choke point for the synthesis of bisected N-glycans. As we know, methods for the construction of β-D-mannosidic linkage include the intramolecular aglycon delivery,7 direct β-D-mannoside coupling protocol,8 and epimerization of β-D-glucopyranosides at the C-2 position by SN2 inversion9 or by sequential oxidation/ reduction routes.10 Some of these methods have been applied to the synthesis of N-glycan oligosaccharides. However, the majority of these approaches need some precious glycosyl donors or tedious protection-deprotection manipulation. In this paper, we have described a convenient procedure to construct the central β-D-mannoside according to Ramstro¨m’s new finding (6) (a) Eller, S.; Schuberth, R.; Gundel, G.; Seifert, J.; Unverzagt, C. Angew. Chem., Int. Ed. 2007, 46, 4173. (b) Schuberth, R.; Unverzagt, C. Tetrahedron Lett. 2005, 46, 4201. (c) Weiss, H.; Unverzagt, C. Angew. Chem., Int. Ed. 2003, 42, 4261. (d) Unverzagt, C.; Seifert, J. Tetrahedron Lett. 2000, 41, 4549. (7) (a) Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994, 72, 1447. (b) Stork, G.; La Clair, J. L. J. Am. Chem. Soc. 1996, 118, 247. (c) Ito, Y.; Ohnishi, Y.; Ogawa, T.; Nakahara, Y. Synlett 1998, 1102. (8) (a) Dudkin, V. Y.; Crich, D. Tetrahedron Lett. 2003, 44, 1787. (b) Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. Tetrahedron Lett. 2003, 44, 1791. (c) Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126, 736. (d) Mandai, H.; Mukaiyama, T. Chem. Lett. 2005, 34, 702. (e) Tanaka, S.; Takashina, M.; Tokimoto, H.; Fujimoto, Y.; Tanaka, K.; Fukase, K. Synlett 2005, 15, 2325. (9) (a) Matsuo, I.; Isomura, M.; Walton, R.; Ajisawa, K. Tetrahedron Lett. 1996, 37, 8795. (b) Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L. X. J. Am. Chem. Soc. 2005, 127, 9692. (10) (a) Shaban, M. A.; Jeanloz, R. W. Carbohydr. Res. 1976, 52, 115. (b) Warren, C. D.; Auge, C.; Laver, M. L.; Suzuki, S.; Power, D.; Jeanloz, R. W. Carbohydr. Res. 1980, 82, 71. (c) Kerekgyarto, J.; Van der Ven, J. G.; Kamerling, J. P.; Liptak, A.; Vliegenthart, J. F. Carbohydr. Res. 1993, 238, 135. (d) Hiro, S.; Usuki, Y.; Iio, H. Carbohydr. Res. 2006, 341, 1796.

on the inversion of carbohydrate hydroxyl groups.11 In the procedure, the designed protecting groups were exactly available for the formation of bisected N-glycans, and all the fragments involved were easily prepared. On the basis of this procedure, octasaccharide A, an N-glycan oligosaccharide of the bisecting type, was synthesized conveniently. Results and Discussion For retrosynthesis of octasaccharide A (Figure 1), it was disconnected to core trisaccharide B, disaccharide donor C, and monosaccharide donor D according to Unverzagt’s strategy for introduction of the bisecting GlcNAc residue.6 R1,3-Arm was first installed followed by the introduction of the bisecting GlcNAc residue, and R1,6-arm was installed in the final glycosylation step. To ensure the introduction sequence, the central β-mannoside in building block B was designed as depicted in Figure 1. The 4,6-O-benzylidene would be removed at a desired stage for the introduction of bisecting GlcNAc via regioselective protection of OH-6. The new structure B would permit good coupling to various building blocks for the synthesis of bisected N-glycans. The retrosynthesis of fragment B is depicted in Figure 2. The intermediate E was designed to prepare B according to Ramstro¨m’s new finding,11 in which the OH-3′′ was acetylated as the neighboring ester group to the triflate. The transformation from E into B was easily performed under the modified Lattrell-Dax reaction conditions by the inversion of OH-23. However, distinguishing OH-3′′ from OH-2′′ with two different ester groups in the β-D-glucopyranoside seemed to be a challenge. In fact, the regioselective protection of one of the two similar secondary 2,3-dihydroxyl groups of β-D-glucopyranoside was difficult to achieve in oligosaccharide synthesis.12 (11) (a) Dong, H.; Pei, Z.; Angelin, M.; Bystroem, S.; Ramstro¨m, O. J. Org. Chem. 2007, 72, 3694. (b) Dong, H.; Pei, Z.; Ramstro¨m, O. J. Org. Chem. 2006, 71, 3306. (12) Varki, A. Glycobiology 1993, 3, 97.

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Wang et al. SCHEME 1.

Synthesis of Trisaccharide B

FIGURE 2. Retrosynthesis of trisaccharide B.

Herein, we explored a new strategy. The key intermediate orthoester F, 3′′-O-chloroacetyl-4′′,6′′-O-benzylidene-R-D-glucopyranose 1′′,2′′-(chloromethyl 3′,6′-di-O-benzyl-2′-deoxy-2′phthalimido-β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy2-phthalimido-β-D-glucopyranosylazide-4′-yl orthoacetate) was designed on the basis of the following considerations: (1) Distinguishing OH-3′′ from OH-2′′ with two different ester groups in the β-D-glucopyranoside could be easily achieved by switching chloroacetyl in F to the acetyl group before 1, 2-orthoester was transformed into the corresponding 3-O-acetyl2-O-chloroacetyl-β-D-glucopyranoside.13 (2) The cyclic 4′′,6′′O-benzylidene protecting group was selected to distinguish OH4′′ and OH-6′′ from OH-3′′ for the introduction of three substituents to the β-mannosyl center. (3) The formation of orthoester F depended critically on the presence of the cyclic 4,6-O-benzylidene protecting groups which stabilized the intermediate oxycarbenium ion.14 (4) The preparation of orthoester F could be conveniently carried out by designing ethyl 4,6-Obenzylidene-2,3-di-O-chloroacetyl-1-thio-β-D-glucopyranoside 1 as the glycosyl donor, which could be easily synthesized from the known compound 3.15 The preparation of the core trisaccharide B started from compound 315 according to the above retrosynthetic analysis (Scheme 1). The unmasked hydroxyls of compound 3 were protected with a chloroacetyl group to afford donor 1, which was treated with disaccharide 216 in the presence of NIS/TfOH (0.1 equiv), giving the desired orthoester 4 (key intermediate F) in 82% yield. The chloroacetyl ester of 4 was removed by using NaOMe, and then the unmasked hydroxyl was acetylated to afford orthoester 5 (95%). In the following attempt to transform orthoester 5 into the corresponding trisaccharide 6, we found that 4,6-O-benzylidene-protected-orthoester 5 was very (13) (a) Ogawa, T.; Beppu, K.; Nakabayashi, S. Carbohydr. Res. 1981, 93, C6. (b) Urban, F. J.; Moore, B. S.; Breitenbach, R. Tetrahedron Lett. 1990, 31, 4421. (c) Wang, W.; Kong, F. J. Org. Chem. 1998, 63, 5744. (14) Yu, H.; Ensley, H. E. Tetrahedron Lett. 2003, 44, 9363. (15) (a) Hou, S. J.; Xu, P.; Zhou, L.; Yu, De Q.; Lei, P. S.; Zou, C. C. Chin. Chem. Lett. 2006, 17, 183. (b) Ennis, S. C.; Gridley, J. J.; Osborn, H. M. I.; Spackman, D. G. Synlett 2000, 11, 1593. (16) (a) Unverzagt, C. Chem. Eur. J. 2003, 9, 1369. (b) Pratt, M. R.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 6149.

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stable even in the presence of 0.5 equiv of TMSOTf at -30 °C. Then we tried TfOH as the promoter, as a result, part of the orthoester 5 was observed to be transformed into trisaccharide 6 in the presence of 0.5 equiv of TfOH at -30 °C after 1 h. When more TfOH was used (1.0 equiv), trisaccharide 6 was afforded in 93% yield. After selective removal of the chloroacetyl group in 6,17 the free hydroxyl group of 7 was inverted in a two-step procedure to afford the desired β-mannoside.11 First, the 2′′-OH of 7 was converted to a good leaving group using triflic anhydride-pyridine and then was substituted by an intramolecular nucleophilic attack of the neighboring acetyl moiety. The SN2 reaction proceeded smoothly by warming the solution of the triflate intermediate in H2O/DMF (1:20) to 50 °C, providing the key fragment B and isomer 8 as a mixture in a very satisfying yield (94%). The ratio of B and 8 was 12.7:1 (determined by 1H NMR analysis), and pure B was obtained after careful column chromatography separation. The anomeric configuration of B was unequivocally deduced from the singlet at 4.85 ppm corresponding to the β-mannosyl anomeric proton and the JC1′′,H1′′ ) 161.8 Hz which is a typical range for β-Dmannopyranoside. By the strategy of formation rearrangement 4,6-O-benzylidene-protected-1,2-orthoester, the core trisaccharide B was synthesized conveniently (62% for 7 steps). The synthesis of disaccharide C is summarized in Scheme 2; the OH-6 of monosaccharide 918 was selectively acetylated to afford 10 (82%). The latter was then treated with donor 1119 in the presence of TMSOTf to afford disaccharide 12 (85%). (17) Nicolaou, K. C.; Caulfield, T. J.; Kataoka, H.; Stylianides, N. A. J. Am. Chem. Soc. 1990, 112, 3693. (18) Baeschlin, D. K.; Green, L. G.; Hahn, M. G.; Hinzen, B.; Ince, S. J.; Ley, S. V. Tetrahedron: Asymmetry 2000, 11, 173. (19) (a) Macmillan, D.; Daines, A. M.; Bayrhuber, M.; Flitsch, S. L. Org. Lett. 2002, 4, 1467. (b) Grundler, G.; Schmidt, R. R. Carbohydr. Res. 1985, 135, 203.

Synthesis of an N-Glycan Octasaccharide SCHEME 2.

Synthesis of Disaccharide Donor C

After cleavage of the biacetal function in 12 (91%), the unmasked hydroxyl was acetylated to afford disaccharide donor C in 96% yield. Monosaccharide D was prepared in five steps from commercially available glucosamine hydrochloride.20 With the core trisaccharide B, disaccharide donor C and donor D in hand, the synthesis of octasaccharide A was then carried out (Scheme 3).6 Treating donor C with trisaccharide B was first performed to install the disaccharide residue on to the OH3′′of central β-mannoside in the presence of NIS/TfOH to afford pentasaccharide 14 in 90% yield (path 1). Compared with the corresponding glycosyl trichloroacetimidate donor,21 thioglycoside donor C proved more effective in the glycosylation. While compound B was not easily separated from the mixture of B and 8. We then tried another route (path 2): After cleaving the acetyl esters of B and 8, the product was treated with disaccharide donor C in the presence of NIS/TfOH. To our delight, the glycosylation reaction preferred the equatorial OH3′′ of benzylidene protected β-mannoside to provide the only product R-(1f3)-linked pentasaccharide,21 in which the OH2′′ of β-mannoside was then acetylated. Pentasaccharide 14 was also provided in 79% yield over 3 steps (Path 2). After cleavage of the benzylidene with TsOH in CH3CN, the OH-6′′ of the β-mannoside was selectively protected with the chloroacetyl group to afford 16. Monosaccharide donor D was treated with 16 to introduce the bisecting residue in the presence of NIS/ TfOH, providing 17 in 78% yield. After selective removal of the chloroacetyl group (91%), installation of the other disaccharide residue onto OH-6′′ of central β-mannoside was finally carried out to provide the bisected N-glycan octasaccharide A in 74% yield. The structure of octasaccharide A was well characterized by 1D NMR and 2D NMR experiments (1H NMR, 13 C NMR, COSY, TOCXY, HMQC, HMBC). (20) (a) Myszka, H.; Bednarczyk, D.; Najder, M.; Kaca, W. Carbohydr. Res. 2003, 338, 133. (b) Silva, D. J.; Wang, H.; Allanson, N. M.; Jain, R. K.; Sofia, M. J. J. Org. Chem. 1999, 64, 5926. (21) Seifert, J.; Unverzagt, C. Tetrahedron Lett. 1996, 37, 6527.

SCHEME 3.

Synthesis of Octasaccharide A

Conclusions In summary, we have developed a new and concise method for the synthesis of bisected N-glycan of the bisecting type based on the 4,6-O-benzylidene-protected-1,2-orthoesters strategy, which greatly simplified the procedure for introduction of the central β-mannoside and assured the sequence for three substituents. Notably, all the fragments involved were easily prepared. Owing to its simplicity and mild reaction conditions, the results of the present investigation will be widely used in the synthesis of N-glycans. Experimental Section 3′′-O-Chloroacetyl-4′′,6′′-O-benzylidene-r-D-glucopyranose 1′′,2′′-(Chloromethyl 3′,6′-Di-O-benzyl-2′-deoxy-2′-phthalimidoβ-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimidoβ-D-glucopyranosylazide-4′-yl Orthoacetate) (4). After a mixture of 1 (8.28 g, 17.80 mmol), 216 (8.78 g, 8.90 mmol), and 4 Å molecular sieves in freshly distilled CH2Cl2 (100 mL) was stirred for 3 h at rt and then cooled to -30 °C, NIS (6.03 g, 26.70 mmol) was added. TfOH (79 µL, 0.89 mmol) was then added dropwise over a 1 min period. The reaction mixture was stirred for 30 min at -30 °C and then filtered through Celite. The filtrate was diluted with CH2Cl2 and washed with aqueousueous NaHCO3, aqueous Na2S2O3, and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (toluene/acetone ) 50:1) to afford 4 (10.1 g, 82%) as a light yellow foam. [R]20D ) +20.8 (c 0.6, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.88-6.75 (m, 33 H), 6.06 (d, 1 H, J ) 5.5 Hz), 5.48 (s, 1 H), 5.44 (dd, 1 H, J ) 9.3, 4.4 Hz), 5.28 (d, 1 H, J ) 8.2 Hz), 5.14 (d, 1 H, J ) 9.9 Hz), 4.86-4.81 (m, 2 H), 4.64 (d, 1 H, J ) 12.1 Hz), 4.56-4.50 (m, 5 H), 4.41 (dd, 1 H, J ) 11.0, 5.5 Hz), 4.31-4.23 (m, 3 H), 4.16-4.12 (m, 2 H), 4.08-4.03 (m, 2 H), 3.96-3.81 (m, 5 H,), 3.70-3.55 (m, 5 H), 3.43 (dd, 1 H, J ) 11.0, 3.3 Hz), 3.37 (d, 1 H, J ) 9.9 Hz), 3.26 (d, 1 H, J ) 9.4 Hz). 13C NMR (150 MHz, 13 C3): δ 166.0, 138.5, 138.3, 138.2, 138.1, 136.5, 129.2, 129.0, 128.3, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.5, 127.5, 127.4, J. Org. Chem. Vol. 74, No. 6, 2009 2511

Wang et al. 127.2, 126.9, 126.1, 125.3, 123.2, 119.6, 101.4, 99.3, 96.6, 85.5, 78.5, 77.7, 76.5, 76.3, 75.4, 75.3, 75.1, 74.5, 74.5, 73.6, 73.1, 72.8, 68.5, 67.7, 67.5, 62.7, 56.5, 55.1, 46.8, 40.5. HRMS (ESI) m/z: calcd for C73H67Cl2N5O19Na [M + Na]+ 1410.3705; found, 1410.3738. 3′′-O-Acetyl-4′′,6′′-O-benzylidene-r-D-glucopyranose 1′′,2′′(Chloromethyl 3′,6′-Di-O-benzyl-2′-deoxy-2′-phthalimido-β-Dglucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimidoβ-D-glucopyranosylazide-4′-yl Orthoacetate) (5). To a solution of 4 (8.33 g, 6.00 mmol) in 1:3 CH2Cl2/CH3OH (400 mL) was added a 1 M solution of NaOMe in MeOH until a pH of 10 was reached. The reaction was stirred for 1 h at rt. Then the solution was neutralized with ion-exchange resin, filtered, and concentrated. The residue was dissolved in CH2Cl2 (100 mL). Pyridine (5 mL), Ac2O (1.2 mL, 12.00 mmol), and DMAP (100 mg) were added. The mixture was stirred for 5 h at rt. One milliliter of methanol was injected into the solution, and the solution was stirred for another 0.5 h at rt. The solution was diluted with CH2Cl2 and washed with aqueous 1 N HCl, saturated aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:2) to yield 5 (7.69 g, 95%) as a white foam. [R]20D ) +22.5 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.83-6.76 (m, 33 H), 6.02 (d, 1H, J ) 5.5 Hz), 5.48 (s, 1 H), 5.34 (dd, 1 H, J ) 9.7, 4.1 Hz), 5.27 (d, 1 H, J ) 8.7 Hz), 5.13 (d, 1 H, J ) 9.2 Hz), 4.86-4.83 (m, 2 H), 4.64 (d, 1 H, J ) 11.9 Hz), 4.56-4.53 (m, 2 H), 4.51-4.48 (m, 3 H), 4.41 (dd, 1 H, J ) 11.0, 5.5 Hz), 4.31-4.27 (m, 2 H), 4.23 (dd, 1 H, J ) 9.6, 8.7 Hz), 4.15-4.12 (m, 2 H), 4.05-4.00 (m, 2 H), 3.87-3.81 (m, 3 H), 3.72-3.65 (m, 3 H), 3.62 (dd, 1 H, J ) 9.5, 9.1 Hz), 3.55 (d, 1 H, J ) 10.6 Hz), 3.42 (dd, 1 H, J ) 11.5, 3.7 Hz), 3.37-3.35 (m, 1 H), 3.29-3.27 (m, 1 H), 1.98 (s, 3 H). 13 C NMR (150 MHz, CDCl3): δ 169.5, 167.7, 138.5, 138.4, 138.3, 138.1, 137.9, 136.8, 134.0, 133.9, 133.7, 131.8, 131.5, 131.2, 129.2, 129.0, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4, 127.1, 126.9, 126.1, 125.3, 123.6, 123.3, 119.5, 101.4, 99.2, 96.6, 85.5, 78.6, 78.2, 76.4, 75.5, 75.1, 74.6, 74.5, 73.6, 73.24, 73.1, 72.8, 68.6, 67.7, 67.6, 56.6, 55.2, 46.9, 20.9. HRMS (ESI) m/z: calcd for C73H68ClN5O19Na [M + Na]+ 1376.4095; found, 1376.4105. O-(3-O-Acetyl-4,6-O-benzylidene-2-chloroacetyl-β-D-glucopyranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-Dglucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimidoβ-D-glucopyranosylazide (6). After a mixture of 5 (1.25 g, 0.92 mmol) and 4 Å molecular sieves in freshly distilled CH2Cl2 (100 mL) was stirred at rt for 3 h and then cooled to -30 °C, TfOH (82 µL, 0.92 mmol) was added dropwise over a 5 min period. The mixture was stirred for 1 h at -30 °C. The reaction mixture was neutralized with Et3N and then filtered through Celite; the filtrate was concentrated and purified by column chromatography on silica gel (toluene/acetone ) 50:1) to afford 6 (1.16 g, 93%) as a white foam. [R]20D ) -15.6 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.90-6.77 (m, 33 H), 5.38 (s, 1 H), 5.27 (d, 1 H, J ) 8.2 Hz), 5.18 (t, 1 H, J ) 9.6 Hz), 5.17 (d, 1 H, J ) 9.2 Hz), 4.96 (dd, 1 H, J ) 9.2, 8.2 Hz), 4.88 (d, 1 H, J ) 12.8 Hz), 4.74 (d, 2 H, J ) 7.7 Hz), 4.66 (d, 1 H, J ) 11.9 Hz), 4.56-4.45 (m, 4 H), 4.39 (d, 1H, J ) 11.9 Hz), 4.28-4.12 (m, 5 H), 4.05 (dd, 1 H, J ) 10.1, 9.6 Hz), 3.93 (d, 1H, J ) 14.7 Hz), 3.89 (d, 1H, J ) 14.6 Hz), 3.65 (d, 1 H, J ) 10.5 Hz), 3.59-3.55 (m, 3 H), 3.44-3.38 (m, 3 H), 3.25-3.20 (m, 3 H), 2.03 (s, 3 H). 13C NMR (150 MHz, CDCl3): δ 170.1, 166.2, 138.4, 138.1, 137.7, 128.7, 128.2, 128.1, 128.0, 127.9, 127.4, 101.5, 99.8, 96.8, 85.5, 78.3, 77.7, 76.4, 75.1, 74.6, 74.5, 73.4, 72.7, 71.8, 68.4, 67.7, 66.8, 66.0, 56.4, 55.2, 40.4, 20.7. HRMS (ESI) m/z: calcd for C73H68ClN5O19Na [M + Na]+ 1376.4095; found, 1376.4034. O-(3-O-Acetyl-4,6-O-benzylidene-β-D-glucopyranosyl)-(1f4)O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosylazide (7). To a solution of 6 (1.79 g, 1.32 mmol) in 80 mL of CH2Cl2/CH3OH (1:3) was added 2,6-lutidine (0.77 mL, 6.60 2512 J. Org. Chem. Vol. 74, No. 6, 2009

mmol) and thiourea (502 mg, 6.60 mmol). After the reaction mixture was stirred for 10 h at rt, it was diluted with CH2Cl2 and washed with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:3) to afford 7 (1.55 g, 92%) as a white foam. [R]20D ) +25.5 (c 0.1, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.84-7.55 (m, 8 H), 7.41-7.26 (m, 15 H), 7.02-6.89 (m, 7 H), 6.81-6.76 (m, 3 H), 5.38 (s, 1 H), 5.28 (d, 1 H, J ) 8.4 Hz), 5.17 (d, 1 H, J ) 9.4 Hz), 5.08 (t, 1 H, J ) 9.5 Hz), 4.86 (d, 1 H, J ) 12.7 Hz), 4.80 (d, 1 H, J ) 12.2 Hz), 4.69 (d, 1 H, J ) 7.7 Hz), 4.61 (d, 1 H, J ) 12.1 Hz), 4.56-4.49 (m, 4 H), 4.41-4.37 (m, 2 H), 4.28 (dd, 1 H, J ) 9.6, 9.2 Hz), 4.22 (dd, 1 H, J ) 11.0, 8.7 Hz), 4.18-4.09 (m, 3 H), 4.06 (dd, 1 H, J ) 10.6, 9.2 Hz), 3.82 (dd, 1 H, J ) 11.5, 2.3 Hz), 3.66 (dd, 1 H, J ) 11.5, 1.9 Hz), 3.58 (d, 1 H, J ) 11.0 Hz), 3.52-3.37 (m, 6 H), 3.33-3.32 (m, 1 H), 3.23-3.19 (m, 1 H), 2.12 (s, 3 H). 13C NMR (150 MHz, CDCl3): δ 171.0, 138.3, 138.1, 137.4, 136.9, 133.8, 131.5, 129.1, 128.6, 128.3, 128.2, 128.0, 127.9, 127.5, 127.3, 127.0, 126.2, 103.6, 101.4, 96.9, 85.6, 78.8, 78.4, 78.0, 76.7, 76.3, 74.9, 74.7, 74.5, 74.4, 74.3, 73.8, 73.3, 72.7, 68.5, 67.7, 67.2, 66.3, 56.5, 55.2, 21.0. HRMS (ESI) m/z: calcd for C71H67N5O18Na [M + Na]+ 1300.4379; found, 1300.4387. O-(2-O-Acetyl-4,6-O-benzylidene-β-D-mannopyranosyl)-(1f4)O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosylazide (B) and O-(3-O-Acetyl-4,6-O-benzylidene-β-D-mannopyranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-βD-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimidoβ-D-glucopyranosylazide (8). To a solution of compound 7 (671 mg, 0.525 mmol) in distilled CH2Cl2 (10 mL) was added pyridine (170 µL, 0.787 mmol). The reaction was cooled to -15 °C. Tf2O (133 µL, 0.788 mmol) was then added dropwise over a 2 min period, and the reaction mixture was stirred for 3 h. The reaction was concentrated under high vacuum. The crude mixture was dissolved in 20 mL of DMF, then 1 mL of H2O was added, and the reaction mixture was stirred for 24 h at 50 °C. Then the reaction mixture was concentrated under high vacuum and purified by flash chromatography (EtOAc/petroleum ether ) 1:3) to afford B and 8 (631 mg, 94%) as a white foam. The ratio of B and 8 was 12.7:1 (determined by NMR analysis). Pure B was obtained by column chromatography on silica gel (300-400 mesh, EtOAc/petroleum ether ) 1:6) and pure 8 was obtained by HPLC (CH3CN:H2O). Compound B. [R]20D ) +0.6 (c 0.2, CHCl3). 1H NMR (600 MHz, DMSO-d6): δ 7.95-6.75 (m, 33 H), 5.61 (s, 1 H), 5.44 (d, 1 H, J ) 6.8 Hz), 5.33 (d, 1 H, J ) 3.7 Hz), 5.30 (d, 1 H, J ) 9.2 Hz), 5.25 (d, 1 H, J ) 8.7 Hz), 4.85 (d, 1 H, J ) 11.4 Hz), 4.85 (s, 1 H), 4.81 (d, 1 H, J ) 12.4 Hz), 4.63 (d, 1 H, J ) 12.4 Hz), 4.56 (d, 1 H, J ) 12.4 Hz), 4.44 (d, 1 H, J ) 11.9 Hz), 4.41 (d, 1 H, J ) 11.9 Hz), 4.38 (d, 1 H, J ) 12.4 Hz), 4.28 (d, 1 H, J ) 12.4 Hz), 4.15-4.01 (m, 5 H), 3.97 (dd, 1 H, J ) 10.6, 8.2 Hz), 3.83-3.74 (m, 3 H), 3.70-3.65 (m, 2 H) 3.63-3.59 (m, 2 H), 3.52 (d, 1 H, J ) 10.5 Hz), 3.39 (dd, 1 H, J ) 11.5, 3.7 Hz), 3.27-3.26 (m, 1 H), 3.20-3.16 (m, 1 H), 2.05 (s, 3 H). 13C NMR (150 MHz, DMSO-d6): δ 169.6, 168.1, 167.4, 167.2, 167.1, 138.4, 138.1, 138.0, 137.9, 137.7, 134.9, 134.8, 130.7, 130.6, 128.3, 128.2, 128.0, 127.9, 127.8, 127.6, 127.5, 127.4, 127.3, 127.2, 126.3, 123.4, 100.9, 98.3, 96.3, 84.9, 78.4, 77.2, 76.3, 76.2, 75.6, 74.9, 74.1, 73.9, 73.7, 72.0, 71.9, 71.8, 68.1, 67.7, 67.6, 66.6, 56.0, 54.6, 20.9. JC1′′,H1′′ ) 161.8 Hz. HRMS (ESI) m/z: calcd for C71H67N5O18Na [M + Na]+ 1300.4379; found, 1300.4337. Compound 8. [R]20D ) +5.3 (c 0.4, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.88 (d, 1 H, J ) 5.5 Hz), 7.79-7.56 (m, 7 H), 7.44-7.41 (m, 2 H), 7.38-7.27 (m, 13 H), 7.30 (d, 2 H, J ) 7.3 Hz), 6.98-6.95 (m, 4 H), 6.93 (d, 1 H, J ) 6.9 Hz), 6.81-6.76 (m, 3 H), 5.43 (s, 1 H), 5.29 (d, 1 H, J ) 8.7 Hz), 5.18 (d, 1 H, J ) 9.6 Hz), 4.92 (dd, 1 H, J ) 10.1, 3.2 Hz), 4.86 (d, 1 H, J ) 12.8 Hz), 4.82 (d, 1 H, J ) 11.9 Hz), 4.79 (s, 1 H), 4.61 (d, 1 H, J ) 11.9 Hz), 4.58-4.50 (m, 4 H), 4.41 (d, 1 H, J ) 12.4 Hz), 4.38

Synthesis of an N-Glycan Octasaccharide (dd, 1 H, J ) 8.7, 1.8 Hz), 4.29 (dd, 1 H, J ) 9.6, 8.7 Hz), 4.25 (dd, 1 H, J ) 10.5, 8.2 Hz), 4.19-4.10 (m, 4 H), 4.06 (dd, 1 H, J ) 10.6, 9.6 Hz), 3.99 (dd, 1 H, J ) 10.1, 9.6 Hz), 3.68 (d, 1 H, J ) 10.1 Hz), 3.60-3.57 (m, 2 H), 3.53 (dd, 1 H, J ) 10.5, 10.1 Hz), 3.44 (d, 1 H, J ) 11.0, 3.2 Hz), 3.40 (dd, 1 H, J ) 10.1, 2.3 Hz), 3.28 (d, 1 H, J ) 9.6 Hz), 3.22-3.18 (m, 1 H), 2.14 (s, 3 H). 13 C NMR (150 MHz, CDCl3): δ 170.2, 138.3, 138.2, 137.5, 137.1, 129.0, 128.6, 128.3, 128.2, 128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.2, 127.0, 126.1, 101.7, 99.8, 96.8, 85.5, 78.3, 77.5, 76.3, 76.2, 75.2, 75.0, 74.6, 74.5, 74.4, 73.4, 72.7, 71.8, 69.3, 68.3, 67.7, 66.8, 56.4, 55.1, 21.0. HRMS (ESI) m/z: calcd for C71H67N5O18Na [M + Na]+ 1300.4379; found, 1300.4340. (2′S,3′S)-Ethyl 3-O,4-O-[2′,3′-Dimethoxybutan-2′,3′-diyl]-6O-acetyl-1-thio-r-D-mannopyranoside (10). To a solution of 918 (13.54 g, 40.0 mmol) in CH2Cl2 (300 mL) was added pyridine (13 mL, 160.0 mmol), and the resulting mixture was cooled to -15 °C. Ac2O (4.7 mL, 50 mmol) was added dropwise over 5 min. The reaction was stirred for 24 h. Subsequently, the solution was diluted with CH2Cl2 and washed with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography on silica gel (EtOAc/ petroleum ether ) 1:1) to afford 10 (12.42 g, 82%) as an oil. [R]20D ) +287.5 (c 0.4, CHCl3). 1H NMR (600 MHz, CDCl3): δ 5.33 (s, 1 H), 4.35-4.31 (m, 2 H), 4.26 (dd, 1 H, J ) 11.9, 5.9 Hz), 4.09 (t, 1 H, J ) 10.1 Hz), 4.02 (dd, 1 H, J ) 3.2, 1.4 Hz), 3.97 (dd, 1 H, J ) 10.4, 2.8 Hz), 3.27 (s, 3 H), 3.22 (s, 3 H), 2.71-2.56 (m, 2 H), 2.07 (s, 3 H), 1.32 (s, 3 H), 1.30 (t, 3 H, J ) 7.3 Hz), 1.28 (s, 3 H). 13C NMR (150 MHz, CDCl3): δ 170.8, 100.4, 100.0, 84.5, 71.0, 68.8, 68.6, 63.5, 62.6, 48.0, 47.8, 25.1, 20.8, 17.7, 17.6, 14.9. HRMS (ESI) m/z: calcd for C16H28O8SNa [M + Na]+ 403.1403; found, 403.1405. (2′S,3′S)-Ethyl 3-O,4-O-[2′,3′-Dimethoxybutan-2′,3′-diyl]-6O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-1-thio-r-D-mannopyranoside (12). A solution of 1119 (3.57 g, 6.16 mmol), 10 (1.95 g, 5.13 mmol), and flameactivated 4 Å molecular sieves were stirred in freshly distilled CH2Cl2 (50 mL) under an Ar atmosphere for 0.5 h at rt. The reaction mixture was then cooled to -30 °C and stirred for 5 min, and then TMSOTf (134 µL, 0.77 mmol) was injected into the solution. The reaction mixture was stirred for 30 min at -30 °C; then the solution was quenched with triethylamine, stirred at rt for 0.5 h, and then filtered through Celite and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:1) to afford 12 (3.48 g, 85%) as a white foam. [R]20D ) +45.2 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.81 (dd, 2 H, J ) 5.0, 2.7 Hz), 7.70 (dd, 2 H, J ) 5.5, 2.8 Hz), 5.83 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.55 (d, 1 H, J ) 8.3 Hz), 5.23 (dd, 1 H, J ) 10.1, 9.2 Hz), 5.12 (s, 1 H), 4.49 (dd, 1 H, J ) 10.6, 8.2 Hz), 4.36 (dd, 1 H, J ) 12.4, 4.1 Hz), 4.21 (dd, 1 H, J ) 12.4, 2.3 Hz), 4.08-4.04 (m, 1 H), 4.03 (dd, 1 H, J ) 2.8, 1.4 Hz), 3.99 (dd, 1 H, J ) 11.9, 1.8 Hz), 3.87-3.84 (m, 2 H), 3.73 (t, 1 H, J ) 10.1 Hz), 3.46 (dd, 1 H, J ) 11.9, 7.3 Hz), 3.20 (s, 3 H), 3.12 (s, 3 H), 2.55-2.44 (m, 2 H), 2.12, 2.03, 1.94, 1.87 (s, 3 H each), 1.19 (t, 3 H, J ) 3.2 Hz), 1.18, 1.14 (s, 3 H each). 13C NMR (150 MHz, CDCl3): δ 170.8, 170.6, 170.2, 169.4, 134.1, 131.6, 129.0, 128.2, 125.3, 123.3, 100.2, 99.5, 96.0, 82.4, 76.3, 72.1, 70.6, 68.7, 67.4, 63.6, 63.0, 61.8, 54.0, 47.9, 47.6, 25.8, 21.4, 20.8, 20.7, 20.6, 20.5, 17.6, 17.4, 14.8. HRMS (ESI) m/z: calcd for C36H47NO17SNa [M + Na]+ 820.2462; found, 820.2487. Ethyl 6-O-Acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimidoβ-D-glucopyranosyl)-1-thio-r-D-mannopyranoside (13). To a solution of 12 (3.48 g, 4.36 mmol) in CH2Cl2 (10 mL) were added 9 mL of TFA and 1 mL of H2O. The reaction mixture was stirred at rt until the starting materials were consumed. Subsequently, the solution was diluted with CH2Cl2 and washed with H2O, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography on silica gel (EtOAc/petroleum ether ) 2:1) to afford 13 (2.70 g, 91%) as a white foam. [R]20D ) +59.4 (c 0.6, CHCl3). 1H NMR (600 MHz,

CDCl3): δ 7.84 (dd, 2 H, J ) 4.6, 3.7 Hz), 7.74 (dd, 2 H, J ) 5.5, 2.7 Hz), 5.81 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.46 (d, 1 H, J ) 8.7 Hz), 5.17 (t, 1 H, J ) 9.6 Hz), 4.91 (s, 1 H), 4.36 (dd, 1 H, J ) 11.0, 8.7 Hz), 4.31 (dd, 1 H, J ) 12.4, 5.0 Hz), 4.19 (dd, 1 H, J ) 12.4, 1.9 Hz), 4.05 (dd, 1 H, J ) 2.3, 0.9 Hz), 4.01 (d, 1 H, J ) 11.4 Hz), 3.93-3.90 (m, 2 H, H-5), 3.86 (dd, 1 H, J ) 11.5, 6.0 Hz), 3.66 (dd, 1 H, J ) 8.7, 6.4 Hz), 3.43 (t, 1 H, J ) 9.6 Hz), 2.82 (d, 1 H, J ) 9.7 Hz), 2.73 (s, 1 H), 2.48-2.40 (m, 2 H), 2.13, 2.05, 2.02, 1.88 (s, 3 H each), 1.14 (t, 3 H, J ) 7.3 Hz). 13C NMR (150 MHz, CDCl3): δ 171.2, 170.7, 170.1, 169.4, 134.3, 131.3, 97.1, 82.0, 80.4, 72.3, 70.9, 70.4, 70.3, 68.7, 68.5, 63.5, 61.7, 60.4, 54.4, 25.3, 21.0, 20.7, 20.6, 20.4, 14.5, 14.2. HRMS (ESI) m/z: calcd for C30H37NO15SNa [M + Na]+ 706.1782; found, 706.1792. Ethyl 3,4,6-Tri-O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-1-thio-r-D-mannopyranoside (C). Compound 13 (1.62 g, 2.37 mmol) and pyridine (1.9 mL, 23.70 mmol) were dissolved in CH2Cl2 (40 mL), and the solution was cooled to 0 °C. Ac2O (1.1 mL, 11.85 mmol) and 100 mg of DMAP were added, and the reaction was stirred for 2 h at rt. Subsequently, the solution was diluted with CH2Cl2, washed with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:1) to afford C (1.74 g, 96%) as a white foam. [R]20D ) +23.3 (c 0.6, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.82 (dd, 2 H, J ) 5.5, 3.2 Hz), 7.72 (dd, 2 H, J ) 5.5, 3.2 Hz), 5.80 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.40 (d, 1 H, J ) 8.7 Hz), 5.16 (dd, 1 H, J ) 10.1, 9.1 Hz), 5.12 (dd, 1 H, J ) 11.0, 10.1 Hz), 4.95 (d, 1 H, J ) 1.4 Hz), 4.89 (dd, 1 H, J ) 10.1, 3.2 Hz), 4.43 (dd, 1 H, J ) 10.6, 8.7 Hz), 4.31 (dd, 1 H, J ) 12.4, 5.0 Hz), 4.27 (dd, 1 H, J ) 3.2, 1.9 Hz), 4.09 (dd, 1 H, J ) 11.9, 2.3 Hz), 4.07-4.04 (m, 1 H), 3.85-3.83 (m, 1 H), 3.76 (dd, 1 H, J ) 12.4, 5.5 Hz), 3.64 (dd, 1 H, J ) 11.9, 2.3 Hz), 2.52-2.42 (m, 2 H), 2.12, 2.04, 2.03, 2.00, 1.99, 1.89 (s, 3 H each), 1.19 (t, 3 H, J ) 7.3 Hz). 13C NMR (150 MHz, CDCl3): δ 170.7, 170.6, 170.4, 170.2, 169.4, 169.2, 134.2, 131.5, 123.4, 96.5, 81.4, 75.9, 72.0, 70.5, 70.4, 68.8, 68.5, 65.9, 62.4, 61.9, 54.3, 25.4, 20.7, 20.6, 20.5, 14.5. HRMS (ESI) m/z: calcd for C34H41NO17SNa [M + Na]+ 790.1993; found, 790.1978. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)O-(2-O-acetyl-4,6-O-benzylidene-β-D-mannopyranosyl)-(1f4)O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-Dglucopyranosylazide (14) Path 1: After a mixture of B (311 mg, 0.243 mmol), C (280 mg, 0.365 mmol), and 4 Å molecular sieves in freshly distilled CH2Cl2 (15 mL) was stirred at rt for 3.0 h and then cooled to -30 °C, NIS (110 mg, 0.486 mmol) was added. TfOH (4 µL, 0.049 mmol) was then added dropwise. The reaction mixture was stirred for 30 min at -30 °C. The reaction mixture was then filtered through Celite, and the filtrate was diluted with CH2Cl2 and washed with aqueous NaHCO3, aqueous Na2S2O3, and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:1) to afford 14 (434 mg, 90%) as a white foam. [R]20D ) +23.4 (c 0.1, CHCl3). 1 H NMR (600 MHz, CDCl3): δ 7.87-6.78 (m, 37 H), 5.46 (dd, 2 H, J ) 10.3, 9.9 Hz), 5.26 (d, 1 H, J ) 8.0 Hz), 5.20 (d, 1 H, J ) 3.7 Hz), 5.16 (d, 1 H, J ) 9.2 Hz), 5.02 (dd, 1 H, J ) 10.3, 9.9 Hz), 4.97 (dd, 1 H, J ) 9.9, 9.1 Hz), 4.94 (s, 1 H), 4.90-4.88 (m, 2 H), 4.84 (dd, 1 H, J ) 10.3, 3.3 Hz), 4.82 (d, 1 H, J ) 11.7 Hz), 4.66 (d, 1 H, J ) 12.1 Hz), 4.53-4.47 (m, 4 H), 4.38 (d, 1 H, J ) 12.1 Hz), 4.34 (d, 1 H, J ) 12.1 Hz), 4.27-4.16 (m, 8 H), 4.04 (dd, 1 H, J ) 10.6, 9.5 Hz), 4.01 (dd, 1 H, J ) 3.3, 1.9 Hz), 3.95 (dd, 1 H, J ) 12.5, 3.7 Hz), 3.85-3.82 (m, 1 H), 3.74 (t, 1 H, J ) 9.5 Hz), 3.71-3.64 (m, 4 H), 3.61-3.55 (m 3 H), 3.49 (t, 1 H, J ) 10.3 Hz), 3.42-3.39 (m, 2 H), 3.21-3.19 (m, 1 H), 3.04-2.99 (m, 1 H), 2.15, 2.05, 2.04, 2.00, 1.98, 1.87, 1.85 (s, 3 H each). 13C NMR (150 MHz, CDCl3): δ 170.6, 170.5, 170.2, 170.1, 169.4, 169.2, 168.5, 167.5, 138.6, 138.5, 138.1, 137.8, 137.3, 134.2, 134.1, J. Org. Chem. Vol. 74, No. 6, 2009 2513

Wang et al. 133.9, 133.8, 131.7, 131.5, 131.4, 130.2, 128.9, 128.7, 128.6, 128.5, 128.3, 128.0, 127.9, 127.7, 127.6, 127.5, 127.2, 127.0, 126.9, 102.4, 98.4, 97.9, 97.1, 95.7, 85.5, 78.7, 78.1, 76.8, 76.6, 76.4, 75.3, 75.2, 74.6, 74.4, 74.3, 73.4, 72.9, 72.8, 71.1, 70.5, 70.4, 69.3, 68.8, 68.5, 68.4, 67.7, 67.4, 66.1, 65.4, 62.8, 61.0, 60.4, 56.4, 55.2, 54.0, 20.8, 20.7, 20.6, 20.5. HRMS (MALDI) m/z: calcd for C103H102N4O35Na [M - N2 + Na]+ 1977.6217; found, 1977.6260. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)O-(2-O-acetyl-β-D-mannopyranosyl)-(1f4)-O-(3,6-di-O-benzyl2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f4)-3,6-di-Obenzyl-2-deoxy-2-phthalimido-β-D-glucopyranosylazide (15). To a solution of 14 (722 mg, 0.364 mmol) in CH3CN (10 mL) was added TsOH · H2O (346 mg, 1.820 mmol). The reaction mixture was stirred for 10 h at rt. Then the solution was neutralized with concentrated Et3N. The crude mixture was diluted with CH2Cl2 and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 1:1) to afford 15 (574 mg, 83%) as a white foam. [R]20D ) -1.7 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.89-6.74 (m, 32 H), 5.73 (dd, 1 H, J ) 10.6, 9.1 Hz), 5.37 (d, 1 H, J ) 8.4 Hz), 5.27 (d, 1 H, J ) 8.5 Hz), 5.16-5.13 (m, 4 H), 4.94 (d, 1 H, J ) 1.8 Hz), 4.89-4.85 (m, 3 H), 4.61 (d, 1 H, J ) 12.1 Hz), 4.54-4.48 (m, 4 H), 4.42-4.38 (m, 3 H), 4.29 (dd, 1 H, J ) 12.5, 5.2 Hz), 4.25-4.09 (m, 7 H), 4.04 (dd, 1 H, J ) 10.6, 9.5 Hz), 3.84-3.78 (m, 4 H), 3.74 (dd, 1 H, J ) 9.9, 9.5 Hz), 3.71 (dd, 1 H, J ) 11.7, 2.9 Hz), 3.63 (d, 1 H, J ) 9.9 Hz), 3.57-3.53 (m, 3 H), 3.43-3.38 (m, 2 H), 3.35 (dd, 1 H, J ) 9.2, 3.7 Hz), 3.23-3.21 (m, 1 H), 3.02-2.99 (m, 1 H), 2.11 (s, 6 H), 2.03, 2.02, 2.02, 1.98, 1.86 (s, 3 H each). 13C NMR (150 MHz, CDCl3): δ 170.8, 170.7, 170.7, 170.2, 170.1, 169.4, 168.5, 167.6, 138.4, 138.1, 137.7, 134.4, 134.2, 134.0, 133.8, 131.7, 131.5, 131.3, 128.7, 128.4, 128.2, 128.1, 127.9, 127.6, 127.5, 127.4, 127.3, 127.0, 123.6, 123.4, 123.2, 98.4, 97.6, 97.1, 97.0, 85.5, 77.6, 77.4, 76.6, 75.4, 75.3, 74.7, 74.5, 74.4, 73.3, 72.9, 71.9, 70.6, 70.4, 69.9, 68.9, 68.3, 67.7, 67.2, 65.4, 62.4, 62.0, 56.4, 55.2, 54.4, 20.9, 20.8, 20.7, 20.6, 20.4. HRMS (MALDI) m/z calcd for C96H98N4O35Na [M - N2 + Na]+ 1889.5904; found, 1889.5905. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)O-(2-O-acetyl-6-O-chloroacetyl-β-D-mannopyranosyl)-(1f4)-O(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-Dglucopyranosylazide (16). Compound 15 (512 mg, 0.270 mmol) and pyridine (0.12 mL, 1.35 mmol) were dissolved in CH2Cl2 (50 mL) and cooled to 0 °C. Chloroacetic anhydride (90%) (61 mg, 0.324 mmol) was added, and the reaction was stirred at 0 °C until compound 15 was consumed. Subsequently, 0.1 mL of methanol was injected into the solution and stirred for 0.5 h at rt. The solution was washed with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 2:3) to afford 16 (447 mg, 84%) as a white foam. [R]20D ) -7.6 (c 0.8, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.85-6.74 (m, 32 H), 5.72 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.38 (d, 1 H, J ) 8.4 Hz), 5.24 (d, 1 H, J ) 8.0 Hz), 5.18-5.13 (m, 4 H), 4.93 (d, 1 H, J ) 1.8 Hz), 4.87-4.84 (m, 3 H), 4.62 (d, 1 H, J ) 12.1 Hz), 4.57-4.46 (m, 5 H), 4.42-4.39 (m, 2 H), 4.36 (d, 1 H, J ) 12.5 Hz), 4.31 (dd, 1 H, J ) 12.1, 4.4 Hz), 4.23-4.08 (m, 8 H), 4.03 (dd, 1 H, J ) 10.6, 9.5 Hz), 3.99 (d, 1 H, J ) 15.0 Hz), 3.96 (d, 1 H, J ) 15.0 Hz), 3.84-3.79 (m, 4 H), 3.64-3.53 (m, 4 H), 3.41-3.34 (m, 3 H), 3.21-3.19 (m, 1 H), 3.15-3.12 (m, 1 H), 3.05 (brs, 1 H), 2.11, 2.10, 2.03, 2.02, 2.02, 1.97, 1.86 (s, 3H each). 13C NMR (150 MHz, CDCl3): δ 170.8, 170.7, 170.2, 170.1, 169.4, 169.4, 168.3, 168.2, 167.6, 138.7, 138.4, 138.1, 137.8, 134.4, 134.0, 133.8, 131.7, 131.5, 131.3, 128.6, 128.3, 128.0, 127.9, 127.6, 127.5, 127.1, 127.0, 126.0, 123.6, 123.3, 123.2, 98.6, 98.1, 97.0, 96.9, 85.5, 78.0, 76.8, 76.7, 76.5, 75.1, 74.6, 74.4, 2514 J. Org. Chem. Vol. 74, No. 6, 2009

74.3, 73.5, 73.2, 72.8, 71.9, 70.6, 70.3, 69.8, 69.0, 68.8, 67.8, 67.7, 67.3, 65.4, 64.4, 62.4, 61.9, 60.4, 56.4, 55.2, 54.3, 40.6, 29.7, 20.8, 20.7, 20.6, 20.4. HRMS (MALDI) m/z: calcd for C98H99ClN4O36Na [M - N2 + Na]+ 1965.5620; found, 1965.5612. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-(1f4)]-O-(2-O-acetyl-6-O-chloroacetyl-β- D mannopyranosyl)-(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2phthalimido-β-D-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy2-phthalimido-β-D-glucopyranosylazide (17). After a mixture of 16 (118 mg, 0.060 mmol), D (135 mg, 0.239 mmol), and 4 Å molecular sieves in freshly distilled CH2Cl2 (10 mL) was stirred at rt for 3 h and then cooled to -30 °C, NIS (68 mg, 0.299 mmol) was added. TfOH (27 µL, 0.299 mmol) was then added dropwise over 1 min. The reaction mixture was stirred for 2 h at -30 °C. The reaction mixture was then filtered through Celite, and the filtrate was diluted with CH2Cl2 and washed with aqueous NaHCO3, aqueous Na2S2O3, and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 3:2) to afford 17 (110 mg, 78%) as a white foam. [R]20D ) -22.9 (c 0.9, CHCl3).1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1 H), 7.87-7.81 (m, 2 H), 7.73-7.66 (m, 7 H), 7.59 (d, 1 H, J ) 7.3 Hz), 7.31-7.24 (m, 7 H), 7.18 (d, 2 H, J ) 6.8 Hz), 7.13 (t, 2 H, J ) 7.8 Hz), 6.94-6.92 (m, 5 H), 6.86-6.85 (m, 3 H), 6.78-6.73 (m, 3 H), 5.76 (dd, 1 H, J ) 11.0, 9.2 Hz), 5.46 (d, 1 H, J ) 8.3 Hz), 5.35 (dd, 1 H, J ) 10.1, 9.6 Hz), 5.28-5.25 (m, 2 H), 5.22 (d, 1 H, J ) 7.7 Hz), 5.13 (d, 1 H, J ) 9.6 Hz), 5.06 (d, 1 H, J ) 3.2 Hz), 4.85-4.79 (m, 3 H), 4.69 (s, 1 H), 4.59 (d, 1 H, J ) 2.8 Hz), 4.57 (s, 1 H), 4.53 (dd, 1 H, J ) 12.4, 3.2 Hz), 4.51-4.41 (m, 6 H), 4.33 (d, 1 H, J ) 12.4 Hz), 4.29 (d, 1 H, J ) 11.9 Hz), 4.27-4.06 (m, 12 H), 4.03-3.97 (m, 2 H), 3.95-3.79 (m, 6 H), 3.73-3.70 (m, 1 H), 3.60 (d, 1 H, J ) 10.5 Hz), 3.53-3.48 (m, 2 H), 3.39-3.33 (m, 3 H), 3.18 (d, 1 H, J ) 9.6 Hz), 3.05-3.04 (m, 1 H), 2.13, 2.09, 2.09, 2.08, 2.05, 2.05, 2.03, 2.01, 1.99, 1.87 (s, 3 H each). 13C NMR (150 MHz, CDCl3): δ 171.0, 170.9, 170.8, 170.5, 170.3, 170.0, 169.6, 169.5, 169.4, 167.3, 138.7, 138.3, 138.0, 137.5, 133.7, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.5, 127.4, 127.1, 127.0, 100.0, 98.7, 97.3, 97.2, 96.9, 85.5, 76.7, 76.5, 75.2, 74.6, 74.5, 74.3, 74.1, 74.0, 73.2, 72.9, 72.7, 72.5, 72.1, 71.4, 71.3, 70.7, 70.3, 69.4, 69.2, 69.0, 68.1, 67.6, 67.0, 65.5, 63.1, 62.4, 61.5, 56.3, 55.4, 55.1, 54.6, 40.5, 20.8, 20.7, 20.6, 20.5. HRMS (MALDI) m/z: calcd for C112H115ClF3N5O44Na [M - N2 + Na]+ 2348.6448; found, 2348.6418. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-(1f4)]-O-(2-O-acetyl-β-D-mannopyranosyl)-(1f4)-O(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-Dglucopyranosylazide (18). To a solution of 17 (78 mg, 0.033 mmol) in methanol (10 mL) were added thiourea (50 mg, 0.66 mmol) and 2,6-lutidine (78 µL, 0.66 mmol). The reaction mixture was stirred for 6 h at 64 °C. Then the solution was concentrated and diluted with CH2Cl2, washed with aqueous 1 N HCl, aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 2:1) to afford 18 (68 mg, 91%) as a white foam. [R]20D ) -23.3 (c 0.1, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.88-6.76 (33 H), 5.78 (dd, 1 H, J ) 10.5, 9.1 Hz), 5.51 (d, 1 H, J ) 8.7 Hz), 5.47 (t, 1 H, J ) 10.1 Hz), 5.30-5.25 (m, 3 H), 5.19 (dd, 1 H, J ) 9.7, 9.6 Hz), 5.15 (d, 1 H, J ) 9.6 Hz), 5.01 (d, 1 H, J ) 3.7 Hz), 4.88 (dd, 2 H, J ) 12.4, 11.0 Hz), 4.81 (dd, 1 H, J ) 10.1, 3.2 Hz), 4.75 (t, 1 H, J ) 9.2 Hz), 4.72 (s, 1 H), 4.61-4.46 (m, 7 H), 4.38 (d, 1 H, J ) 11.9 Hz), 4.34 (s, 1 H), 4.25-3.99 (m, 12 H), 3.85-3.73 (m, 5 H), 3.62 (dd, 2 H, J ) 9.6, 9.2 Hz), 3.55-3.50 (m, 2 H), 3.43-3.38 (m, 3 H), 3.30 (dd, 1 H, J ) 9.6, 3.2 Hz), 3.20 (d, 1 H, J ) 10.1 Hz),

Synthesis of an N-Glycan Octasaccharide 2.67 (d, 1 H, J ) 9.6 Hz), 2.13, 2.12, 2.07, 2.06, 2.05, 2.01, 2.01, 1.99, 1.98, 1.87 (s, 3H each). 13C NMR (150 MHz, CDCl3): δ 172.4, 171.2, 171.0, 170.8, 170.4, 170.3, 169.8, 169.6, 169.5, 168.5, 167.6, 167.3, 157.2, 157.0, 138.6, 138.4, 138.1, 137.3, 134.2, 134.1, 133.8, 131.6, 131.5, 131.3, 128.6, 128.4, 128.3, 128.0, 127.9, 127.6, 127.5, 127.0, 126.4, 123.8, 123.4, 123.2, 116.8, 114.9, 101.0, 98.4, 97.1, 97.0, 96.9, 85.5, 76.8, 76.6, 76.5, 76.3, 75.4, 74.7, 74.5, 74.3, 74.2, 73.6, 73.4, 72.9, 72.5, 72.4, 72.0, 71.3, 71.0, 70.5, 21.0, 20.9, 20.7, 20.6, 20.5, 20.3. HRMS (MALDI) m/z: calcd for C110H114F3N5O43Na [M - N2 + Na]+ 2272.6732; found, 2272.6703. O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r-D-mannopyranosyl)-(1f3)[O-(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-(1f4)]-[O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-βD -glucopyranosyl)-(1f2)-O-(3,4,6-tri-O-acetyl-r- D mannopyranosyl)-(1f6)]-O-(2-O-acetyl-β-D-mannopyranosyl)(1f4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β- D glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimidoβ-D-glucopyranosylazide (A). After a mixture of 18 (80 mg, 0.035 mmol), B (54 mg, 0.070 mmol), and 4 Å molecular sieves in freshly distilled CH2Cl2 (10 mL) was stirred at rt for 3 h and then cooled to -30 °C, NIS (24 mg, 0.105 mmol) was added. TfOH (3 µL, 0.035 mmol) was then added. The reaction mixture was stirred for 1 h at -30 °C. The reaction mixture was then filtered through Celite, and the filtrate was diluted with CH2Cl2 and washed with aqueous NaHCO3, aqueous Na2S2O3, and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel (EtOAc/petroleum ether ) 2:1) to afford A (78 mg, 74%) as a white foam. [R]20D ) -13.3 (c 0.1, CHCl3). 1H NMR (600 MHz, CDCl3): δ 8.32 (d, 1 H), 7.88-6.72 (m, 36 H), 5.79 (dd, 1 H, J ) 10.5, 9.1 Hz), 5.50 (d, 1 H, J ) 8.2 Hz), 5.42 (dd, 1 H, J ) 10.6, 9.2 Hz), 5.35 (dd, 1 H, J ) 10.1, 9.7 Hz), 5.30-5.25 (m, 2 H), 5.19 (d, 1 H, J ) 8.2 Hz), 5.16 (dd, 1 H, J ) 9.7, 9.6 Hz), 5.12 (dd, 1 H, J ) 10.1, 9.6

Hz), 5.11 (d, 1 H, J ) 9.6 Hz), 5.01 (d, 1 H, J ) 3.2 Hz), 4.96-4.93 (m, 2 H), 4.89-4.84 (m, 3 H), 4.74 (s, 1 H), 4.61-4.53 (m, 6 H), 4.49 (dd, 1 H, J ) 10.5, 8.2 Hz), 4.46 (s, 1 H), 4.44 (d, 1 H, J ) 10.5, 8.2 Hz), 4.40 (d, 1 H, J ) 12.8 Hz), 4.35 (d, 1 H, J ) 12.4 Hz), 4.31 (s, 1 H), 4.26 (dd, 1 H, J ) 10.6, 8.3 Hz), 4.21 (d, 1 H, J ) 11.9 Hz), 4.16-4.09 (m, 9 H), 4.06-3.99 (m, 6 H), 3.92-3.86 (m, 3 H), 3.82-3.77 (m, 3 H), 3.74 (dd, 1 H, J ) 12.4, 2.8 Hz), 3.67 (d, 1 H, J ) 11.0 Hz), 3.57-3.55 (m, 2 H), 3.46-3.42 (m, 3 H), 3.34-3.26 (m, 3 H), 3.20 (d, 1 H, J ) 10.1 Hz), 2.91 (dd, 1 H, J ) 9.6, 2.8 Hz), 2.14, 2.14, 2.09, 2.08, 2.06, 2.05, 2.02, 2.02, 2.01, 2.00, 1.99, 1.88, 1.88, 1.87, 1.85 (s, 3 H each). 13C NMR (150 MHz, CDCl3): δ 171.7, 170.9, 170.9, 170.8, 170.6, 170.3, 170.2, 169.9, 169.6, 169.4, 169.2, 169.2, 167.9, 167.3, 167.0, 138.5, 138.4, 138.0, 137.5, 134.3, 134.0, 133.8, 133.6, 131.8, 131.5, 131.3, 128.9, 128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6, 127.5, 127.4, 127.0, 123.8, 123.5, 123.1, 100.5, 98.5, 98.0, 97.7, 97.0, 97.0, 96.5, 85.4, 78.3, 76.7, 75.5, 75.3, 74.5, 74.4, 74.2, 73.8, 73.5, 73.4, 73.2, 72.7, 72.2, 71.4, 71.2, 71.0, 70.8, 70.4, 70.3, 69.9, 69.8, 69.1, 68.9, 68.4, 68.2, 67.4, 67.3, 66.8, 66.6, 65.7, 63.8, 62.9, 62.5, 61.4, 61.4, 56.3, 55.1, 54.9, 54.7, 54.2, 20.9, 20.7, 20.6, 20.5, 20.4. HRMS (MALDI) m/z: calcd for C142H149F3N6O60Na [M - N2 + Na]+ 2977.8637; found, 2977.8623.

Acknowledgment. This work is supported by the National Basic Research Program of China (2003CB716403). Supporting Information Available: General methods, the synthesis of compounds 1, D, and 14 (path 2), 1H NMR and 13 C NMR spectra for compounds 1, 4-8, 10, 12-18, and A-D. This material is available free of charge via the Internet at http://pubs.acs.org. JO900016J

J. Org. Chem. Vol. 74, No. 6, 2009 2515